5 DR Roeland Wouters - Una Aproximación Integral Al Cultivo Larvario Con Enfoque en La Nutrición y Calidad de Postlarvas

AN INTEGRATED LARVAL SHRIMP REARING APPROACH
WITH
FOCUS ON NUTRITION AND PL QUALITY:
Stretching the balance between
experimental feed and Artemia
UNA PROXIMACIÓN INTEGRAL AL CULTIVO LARVARIO CON ENFOQUE EN LA

NUTRICIÓN Y LA CALIDAD DE LAS POST-LARVAS:
En búsqueda de un nuevo balance entre dietas experimentales y Artemia
Roeland Wouters
D. Grymonpre, C. De Maesschalck,
P. Joos, V. Calabuig, P. Rattayaporn, A. Rekecki,
E. Naessens, G. Rombaut, P. Lavens
Wednesday 25 October - 12:20 – 13:00

Larval shrimp rearing – Nutrition and PL quality
Introduction
1. Disease > 2. PL quality & availability > 4. Feed cost > 10. Feed quality
2
Introduction
1. Feed cost > 2. Disease > 6. PL quality & availability > 9. Feed quality
3
Ecuador hatcheries
4
Introduction
Holistic approach
PL QUALITY
Introduction
• Survey on PL Quality
E. Werbrouck et al. 2017
Introduction
• Robust PL’s: Why?
– Postlarval quality and its long-‐term effects have grown in interest to hatchery and
grow-‐out farmers as the shrimp industry faces challenges overcoming diseases, while
moving towards greater integration, genetic improvement and increased control.
• Robust PL’s: How

– Improved larval rearing techniques
• Nutrition
§ Innovative feeds that promote survival, growth and long-‐term PL quality
§ Decreased dependence on Artemia cysts
• Health
§ Boost stress resistance and resilience
§ Microbial management / Reduce Vibrio load
Shrimp hatchery facilities at ITARC

24x 3MT tanks
Set1: 60x 175L tanks Series of

P. vannamei
larval trials
(N5-‐PL10)
Set2: 60x 175L tanks

Improved Nutrition
• Best Balance
– Best Balance between live food and formulated feed FRIPPAK Fresh GOLD
• Reduction of Artemia cyst consumption by factor 2
Possible Artemia reduction (with FRIPPAK FRESH Gold):

normal, decreased and zero Artemia cyst use
Best
Balance
Relative performance (%)
100 Survival
80 Dry Weight
60
40
20
0
3 1,5 0
Artemia use (kg cysts/Mio fry)
Improved Nutrition
Best Balance
Nursery growth after Artemia replacement

in the Hatchery phase
% Artemia replacement
(ar) in hatchery phase
Improved Nutrition
• Further stretching the balance
– A slow gradual decrease in Artemia consumption statistics demonstrates that hatcheries
world-wide learned to reduce their dependence on live food
– Hatcheries that have implemented Artemia reduction themselves have a stronger believe
in low Artemia consumption protocols
yes no
Do you think that
feeding regimes Americas
with reduced
0 20 40 60 80 100
Artemia
consumption can
produce good SE Asia
postlarvae quality?
Improved Nutrition
Contribution of Artemia
Effect of Artemia replacement in late PL
– Trial demonstrated the negative effect of stopping Artemia feeding in PL
stages
Growth
800
700
Dry Weightat PL10 (µg)
600
500
400
300
200
100
0
Stop PL8 Stop PL7 Stop PL6 Stop PL5 Stop PL4 Stop PL3
Stage at wich Artemia feeding stopped
ITARC C166
Improved Nutrition
Contribution of Artemia
Artemia nauplii are more efficiently assimilated than formulated feeds
ITARC C244
75% of
Total Feeding:
280µg DM
25% Artemia nauplii contributes more to larval growth and survival than 75% dry feed
Results confirmed by study J. Gambao-Delgado & L. Levay (2009), Aquaculture 297

Improved Nutrition
Growth
– As growth is mainly requiring protein synthesis, the focus in the current study
was put on protein nutrition
– Earlier published studies with protein hydrolysate in diets for marine fish and
shrimp larvae revealed the importance of amino acid balance.
– Presence of different proteases during larval development suggests age-‐
specific digestive capacities.
Protein Nutrition
Amino acid composition
Peptide size
Digestibility
6/11/17
Protein nutrition
Peptide molecular weight distribution
à Free amino acids, dipeptides and tripeptides are readily absorbed by
enterocytes and enter the organism at higher rates than larger proteins.
Size Exclusion
Chromatography (SEC)
SEC-‐screening of
ingredients can clearly
distinguish three groups
based on protein size
Protein nutrition
MW differences give the opportunity to formulate diets with
different protein profiles
Protein nutrition
in vitro digestibility assay for P. vannamei larvae and PL’s
(pH-‐stat determination of the degree of protein hydrolysis, %DH)
à Efficient feed formulation, processing and feeding practices

+ suitable absorption/utilization of balanced amino acid pools
à New in vitro protocol developed in collaboration with Lemos & Yasumaru
(University of São Paulo), using enzymes extracted from larval and postlarval
P. vannamei
Protein nutrition
in vitro protein digestibility with P. vannamei enzyme extract
digestibility
14
13
12
Degree of hydrolysis (%)
11
10
9
8
7
6
5
4
3
2
1
0
casein gelatin fish meal squid meal freeze-‐dried soy bean wheat gluten brewers
fish meal yeast
Z2 M2 PL3
à Protein digestibility increased during larval development and reached maximum at
Mysis 2 stage, followed by a decrease from Mysis 2 to PL3
à Vegetable terrestrial proteins were less digestible than marine animal proteins
à Distinction between animal marine protein sources & effect of processing
à Rapid screening of ingredients for the production of highly-‐digestible larval aquafeeds
Protein nutrition
Shrimp trial with feed containing highly-‐digestible, low molecular-‐weight,
water-‐soluble peptide source (EF-‐PQ)
Peptide MW distribution
15 à Experimental feed (EF) with
% of soluble protein
10 inclusion of an ingredient

selected on Protein Nutrition /
5
Protein Quality (PQ)
0
EF-‐CON EF-‐PQ
>20 kDa 20-‐10 kDa 10-‐5 kDa 5-‐1 kDa <1 kDa
Survival
Growth à Increased survival and growth
70 800
ITARC C236 700
à EF-‐PQ enabled drastic
60
reduction of Artemia
Survival Z1-‐PL10 (%)
600
Dry Weight PL10 (µg)
50
40
500 consumption
400
30
300
20
200
10 100
0 0 0.5kg cysts consumed per M PL10
EF-‐CON_0.5 EF-‐PQ_0.5 EF-‐CON_0.5 EF-‐PQ_0.5
Improved Nutrition
Growth (individual Dry Weight)
– Innovative feed development was obtained by combining new insights in protein
quality with adapted feed technology.
– This technology was required to control the leaching of low-MW water-soluble
nutrients such as amino acids, di- and tripeptides.
ITARC C246
1000
900
Protein Q
Technology
800
700
Dry Weight (µg)
600
500
400
300
200
100 1 kg cysts
0 +
EF-‐A_1 EF-‐B_1 EF-‐C_1 EF_C
+
live algae
Innovative feed performance
Trial 1 Trial 2
Growth ITARC 17LS01 Growth

800 1000
avg

603µg
800
600
avg 600
338µg
400
400
200 200 2.35kg

cysts 0.46kg
2.34kg 0.52kg 0.41kg
0 0
COMM_2,34 COMM_0,52 EF-‐C_0,41 COMM_2,35 EF-‐C_0,46
Survival Survival Survival Survival Survival
69% 62% 76% 66% 68%
Repeatedly good survival and good growth results with newly developed
feed EF-C, applying very low Artemia cyst consumption (less than 0.5kg)
and adjusted feeding strategy.
Innovative feed performance

Follow-up trial in nursery tanks:
Trial 3 both treatments received same feed
during nursery culture
ITARC 17LS03r
1000
Growth
16000

800
12000
600
400 8000
200 2.46kg
cysts 0.45kg 4000
0
COMM_2,46 EF-‐C_0,45
0
Survival Survival COMM_2,46 EF-‐C_0,45
58% 65%
Good results with newly developed feed

EF-‐C are continued in the nursery phase
Feed EF-‐C produced larger/stronger PL’s
Larval shrimp rearing – Nutrition, Health and PL quality
Improved Nutrition & Health

Holistic approach
FRY QUALITY
Larval shrimp rearing – Health and PL quality
Improved Health
• A strong believe in the role of health products
– The survey reflects the strong believe of hatchery managers in the benefit of health
products for producing stronger PL’s and reducing the risk of disease outbreak:
Vibrio suppressors, Health booster feeds, Probiotics
Do you think that …
1. microbial management in the

hatchery influences PL quality?
2. there is a link between poor PL

quality and the incidence of disease?
Improved Health
Vibrio suppressors / Increased robustness
Specific phytobiotic mix PL transport trial
Vibrio load after 24h transport (PL10)

1000000
Vibrio sp.
100000
Vibrio
10000 parahaemolyticus
1000
100
10
Control FIT Control FIT
Postlarvae Water
Improved Health
Vibrio suppressors / Increased robustness
Specific phytobiotic mix
PL10 Survival after 43°C

temperature stress
Gene expression in PL10 stage shrimp
60 3
50 2
40 Control
1
30 FIT
20 0
HSP70 Crustin1
10
0 Upregulation of HSP70 RNA and Crustin1 RNA

after 1h after 2h
Control FIT
Improved Health
Vibrio suppressors
Applies selected plant extracts (incl. Berberis) to specifically suppress
Vibrio development during the hatching process
Bacterial counts in HATCHING WATER (24h) of GSL cysts
1.E+08 1.E+08
1.E+06 1.E+06
CFU/ml
CFU/ml
1.E+04 1 year storage 1.E+04
1.E+02 1.E+02
1.E+00 1.E+00
MA TCBS MA TCBS
Not treated cysts Not treated cysts

Berberis treatment Berberis treatment
SPECIFIC action on VIBRIO’S Bacterial reduction is STABLE

during at least ONE YEAR
Improved Health
Vibrio suppressors
Bacterial counts on HATCHED NAUPLII (24h) of GSL cysts
1.E+05
1.E+04
100 x
reduction
CFU/ml
1.E+03
1.E+02
1.E+01
1.E+00
MA TCBS
Not treated cysts

Berberis treatment
Artemia nauplii
Improved Health
Probiotics Health booster feeds

Positive effect on PL5 stage Positive effect on PL10
survival and growth stress resistance
PL10 Survival (%) to a

salinity stress test
Stress test survival (%)

100
80
60
40
20
0
Feed only Feed + S-‐PAK
PL quality through improved nutrition and health
• Summary
– Growing shrimp industry requires robust / high-quality postlarvae
– PL quality can be promoted through improved larval rearing techniques
– Innovative feeds have been developed based on new feed technology and
continued efforts in the field of ingredient digestibility and protein nutrition
– These new feeds promote survival, growth and PL quality, also under low
Artemia consumption protocols
– Health practices help producing robust and healthy PL’s, hence assist in
reducing mortalities during disease incidence in the farm
Larval shrimp rearing – PL quality & Grow-out performance
Improved PL quality à Improved grow-out performance
“Long-term stress and handling

stress negatively affected growth,
molting and feed consumption in L.
vannamei juveniles.”
(Mercier et al. 2009)
Higher survival to a PL stress test
is associated to:
• “greater survival during
stocking”
(Fegan 1992;; Racotta et al. 2004)
• “better survival and growth
during grow-out”
(Aquacop et al. 1991)
Grow-‐out trial (Ecuador):

• PL’s originated from the same hatchery received two different larval feeding
regimes (A & B) à 12 shipments, 8.5 million PL à stocked in 0.3 ha ponds at
the grow-‐out farm and harvested after 45 days
Pond survival
67
80 A A = control hatchery feeding regime
44
Survival (%)
60 B B = regime with microencapsulated larval feeds

40
20
0
Hatchery diet
An analysis of the survival of hatchery reared Penaeus vannamei, Boone, larvae.
H. Lucien-‐Brun, A. Aguilar, J.A. Salvador and Daniel O’C Lee

Experiment (CENAIM, Ecuador): Growth increased
... with 17% with PL’s that better resisted osmotic stress test (OST)
... with 19% with PL’s that could swim counter-‐current
Juvenile growth
Relative growth increase (%)

120
CCT 110
Counter-‐current test (CCT) 100

High Quality PL
90 Regular PL
OSS
CCT
PL quality evaluation
Naessens et al. 1995
• Effect of stocking high quality PL’s in grow-out ponds

Effects in the first month after pond stocking
– improved survival rate and increased growth
– reduced transfer of pathogens to the ponds
– allows a good initial count of the number of PL stocked
– improved control over juvenile stocking densities à more accurate
calculation of feeding quantities
Long-term effects during grow-out
– improved survival rate, increased growth, better FCR, better disease
resistance
– more accurate calculation of feeding quantities
è increased final pond crop yield
Assumed cost benefit of stocking improved PL quality
Hypothetical cost benefit by stocking better quality postlarvae:
Shrimp grow-‐out pond (1 ha) Regular Improved Difference
stocked with 1 Mio PL’s PL quality PL quality (%)
(harvest 15 g shrimp)
Survival transport (%) 91 95 +4
Survival pond (%) 75 80 +7
Growth rate (g/wk) 1,21 1,25 +3
Culture periods (days) 89 85 -‐4
Feed cost (USD) 24413 23687 -‐3
Additional costs (USD) 19207 19207 0
Total cost (USD) 43620 42888 -‐2
Cost/kg shrimp (USD) 2,84 2,43 -‐14
Yield/pond (USD) 76795 88343 +15
Profit/pond (USD) 33175 45455 +37
PL quality through improved nutrition and health
• Summary
– Growing shrimp industry requires robust / high-quality postlarvae
– PL quality can be promoted through improved larval rearing techniques
– Innovative feeds have been developed based on new feed technology and
continued efforts in the field of ingredient digestibility and protein nutrition
– These new feeds promote survival, growth and PL quality, also under low
Artemia consumption protocols
– Health practices help producing robust and healthy PL’s, hence assist in
reducing mortalities during disease incidence in the farm
– Stocking high quality PL’s in grow-out ponds has proven beneficial effect
on survival and growth in the weeks after pond stocking
– Assumed long-term effects of stocking high quality PL are increased crop
yield and cost benefit for the grow-out farmer
Take-away-message
Do not compromise hatchery performance by cutting costs on
necessary feeds, health products and microbial management.
Instead, invest in optimizing PL quality from the start of hatchery and
get higher net return at pond harvest.
Thank you!

5 DR Roeland Wouters - Una Aproximación Integral Al Cultivo Larvario Con Enfoque en La Nutrición y Calidad de Postlarvas

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AN INTEGRATED LARVAL SHRIMP REARING APPROACH

UNA PROXIMACIÓN INTEGRAL AL CULTIVO LARVARIO CON ENFOQUE EN LA

Wednesday 25 October -­ 12:20 – 13:00

• Robust PL’s: How

Shrimp hatchery facilities at ITARC

Set1: 60x 175L tanks Series of

Set2: 60x 175L tanks

Possible Artemia reduction (with FRIPPAK FRESH Gold):

Nursery growth after Artemia replacement

Results confirmed by study J. Gambao-Delgado & L. Levay (2009), Aquaculture 297

Amino acid composition

à Efficient feed formulation, processing and feeding practices

10 inclusion of an ingredient

Innovative feed performance

Growth ITARC 17LS01 Growth

Dry Weight PL10 (µg)

200 200 2.35kg

Innovative feed performance

Dry Weight PL23 (µg)

Good results with newly developed feed

Improved Nutrition & Health

Vibrio suppressors, Health booster feeds, Probiotics

Do you think that …

1. microbial management in the

2. there is a link between poor PL

Vibrio load after 24h transport (PL10)

PL10 Survival after 43°C

0 Upregulation of HSP70 RNA and Crustin1 RNA

Bacterial counts in HATCHING WATER (24h) of GSL cysts

1.E+04 1 year storage 1.E+04

Not treated cysts Not treated cysts

SPECIFIC action on VIBRIO’S Bacterial reduction is STABLE

Bacterial counts on HATCHED NAUPLII (24h) of GSL cysts

Not treated cysts

Probiotics Health booster feeds

PL10 Survival (%) to a

Stress test survival (%)

PL quality through improved nutrition and health

Improved PL quality à Improved grow-out performance

“Long-term stress and handling

Improved PL quality à Improved grow-out performance

Grow-­‐out trial (Ecuador):

60 B B = regime with microencapsulated larval feeds

Improved PL quality à Improved grow-out performance

Relative growth increase (%)

Counter-­‐current test (CCT) 100

Improved PL quality à Improved grow-out performance

• Effect of stocking high quality PL’s in grow-out ponds

PL quality through improved nutrition and health

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Wednesday 25 October - 12:20 – 13:00

Grow-‐out trial (Ecuador):

Counter-‐current test (CCT) 100